The molecular basis for the tissue-specific expression of interleukin 4 (IL-4) in Th2 cells has begun to be elucidated. We have recently shown that the provision of three distinct proteins, the c-maf protooncogene, the transcription factor Nuclear Factor of Activated T cells (NFAT), and a novel nuclear antigen, NFAT-Interacting Protein 45 kD (NIP45), confers on a non-T cell the ability to produce endogeneous IL-4. We demonstrated that NIP45 synergized potently with all NFAT family members to transactivate an NFAT reporter gene, and that it synergized with NFATp and c-maf to activate the IL-4 promoter. More important, this synergy was so pronounced that B lymphoma cells produced levels of endogenous IL-4 that approximated those produced by primary Th2 cells, upon transient overexpression of NIP45 with NFATp and c-maf. NIP45 was essential for the production of significant amounts of IL-4: the provision of c-maf and NFAT alone resulted in very small amounts of IL-4. Given its clear biologic relevance for IL-4 production, we wish to address the many questions that remain to be answered about the function of NIP45. We cloned NIP45 by virtue of its interaction with NFAT, but we do not know the mechanism by which it augments maf/NFAT-dependent IL-4 production. One exciting possibility is that it is a new class of coactivator protein (Aim 1). We also do not yet know whether NIP45, acting in concert with NFAT, can alter the production of cytokines other than IL-4 or can affect the transcription of other yet-to-be-identified NFAT-target genes. The search for NFAT/NIP45-inducible genes (Aim 2), and the production and characterization of a NIP45-deficient mouse (Aim 3) should help provide insights. We know that NFAT is one partner for NIP45, and we have preliminary data that another RHD factor, NFkappaB p65, also binds NIP45. As yet, we do not know if this latter interaction is functionally important for endogeneous gene expression or whether it holds for other NF-kappaB family members (Aim 4). If so, then the potential number of NIP45 regulated genes becomes much larger. Finally, NIP45 might interact with other proteins, possibly transcription factors or proteins important in signal transduction from the membrane to the nucleus. The isolation of novel NIP45-interacting proteins proposed in Aim 5 should clarify these pathways.